Epicardial slices: an innovative 3D organotypic model to study epicardial cell physiology and activation

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Experimental design

This study aimed to develop an ex vivo 3D organotypic model of the epicardial/myocardial interface, which would enable studies directed at identifying mechanisms of adult epicardium reactivation. Our experiments verified the maintenance of the tissue architecture in the slices and the preservation of a living and healthy epicardial cells monolayer and myocardial tissue. Following Tβ4 stimulation, we evaluated in situ the upregulation of epicardial embryonic genes, and the EMT, migration and differentiation of the WT1+ cells.

Tissue samples

Swine hearts were obtained from 4–6 weeks old piglets, from The Pirbright Institute, (Pirbright, UK), and 16–20 weeks old pigs from the Newman’s Abattoir (Farnborough, UK). Animal experiments were carried out under the Home Office Animals (Scientific Procedures) Act (1986) (ASPA) and approved by the Animal Welfare and Ethical Review Board (AWERB) of The Pirbright Institute. The animals were housed in accordance with the Code of Practice for the Housing and Care of Animals Bred.

Pigs of 4–6 weeks were euthanized by an overdose of 10 ml pentobarbital (Dolethal 200 mg/ml solution for injection, Vetoquinol UK Ltd). All procedures were conducted by Personal License holders who were trained and competent and under the Project License PPL70/8852. After exsanguination, the thorax was opened using a sterile scalpel, and a transversal incision of the sternum allowed to open the chest and access the mediastinum. The hearts were rapidly collected after transection of the great vessels, maintaining the pericardium membrane intact, and then immediately submerged in 300 ml of ice-cold cardioplegia solution23 (NaCl 110 mM; CaCl2 1.2 mM; KCl 16 mM; MgCl2 16 mM; NaHCO3 10 mM; pH 7.4), to remove the excess of blood. Retrograde heart perfusion (Fig. 1, step 1) was performed within 1 min from the excision using a 100 ml sterile syringe full of ice-cold cardioplegia connected to a three-way valve equipped with a 0.5 cm luer. The luer was inserted in the aorta and secured in position with a nylon cable tie. The cardioplegia was slowly injected into the heart, any bubble in the solution was removed by revolving the valve to direct the flow towards the open end. Effective flushing of the residual blood from the heart vessels was verified visually, before opening the pericardial membrane. Ventricles were removed by cutting along the left anterior descending artery and the posterior descending artery and immersed in ice-cold cardioplegia.

Abattoir pigs were euthanized according to local regulations. Abattoir pigs’ hearts were harvested from farm pigs at the abattoir and immediately washed with 500 ml of ice-cold cardioplegia, on-site. Ventricles were removed cutting along the left anterior descending artery and the posterior descending artery and then put in a hermetic plastic box with fresh ice-cold cardioplegia (800–1000 ml).

Independently from the source, samples submerged in ice-cold cardioplegia were packed in an insulated polystyrene box filled with cooler packs and transported to the lab. Tissue was only removed from the solution at the time of cutting (1–3 h from collection).

Slice preparation

Before the epicardial slice preparation procedure may commence, the following needs to be completed: preliminary vibratome set up, preparation of embedding solution, embedding area staging, and recovery bath assembling.

The high precision vibratome (Leica, VT1200S) used for sectioning the tissue was cleaned with 70% ethanol and then distilled water. A double edge razor blade (Wilkinson Sword) was mounted onto the blade holder. Once in place, we checked the optimum positioning of the blade using Leica’s VibroCheck according to the manufacturer’s instructions. This allowed to minimize z-axis deflection of the blade during cutting at values comprised between −0.2 and 0.2 µm, avoiding tissue damages. The cutting amplitude was set at 1.5 mm and speed at 0.03 mm s−1. Next, the vibratome bath was mounted and filled with cold cutting/recovery solution (NaCl 140 mM; CaCl2 1.8 mM; KCl 6 mM; MgCl2 1 mM; Glucose 10 mM; 2-[4-(2-hydroxyethyl)piperazin-1-yl] ethanesulfonic acid (HEPES) 10 mM; 2,3-Butanedione monoxime (BDM) 10 mM; pH 7.4, 4 °C). The solution was bubbled with 99.5% oxygen for at least 30 min before starting to cut, and the outer part of the bath was filled with ice to maintain the temperature in the specimen bath.

The tissue embedding solution was prepared by dissolving 5% w/v of low melting agarose (ThermoScientific, TopVision Low Melting Point Agarose) in cutting/recovery solution, and microwaving briefly until completely melted. The embedding solution was left to cool down in a water bath set at 37 °C. The compliant surface for the alignment of the epicardial blocks consisted of a 0.5 cm thick agarose cushion. The cushion was made by dissolving 2% w/v of agarose (Invitrogen UltraPure Agarose) in a cutting/recovery solution, the liquid mix was poured into a Petri dish and allowed to solidify at room temperature before being placed on ice. For the tissue dissection and embedding area we used: a polystyrene box full of ice, a petri dish, and the agarose cushion already prepared, organized as in Fig. 1, step 2. Other necessary equipment were: single edge steel blades, anatomical forceps, 3D printed plastic ring, plastic Pasteur pipettes, and cyanoacrylate glue.

The recovery bath for the epicardial slices was prepared by placing a six-well culture plate with pierced bottom in a large plastic box filled with recovery solution at room temperature. In each well was placed a cell culture insert, and the cutting/recovery solution was bubbled with 99.5% oxygen continuously.

Once the low melting agarose solution was cooled at 37 °C, the dissecting area was prepared and the agarose cushion cooled down, it was possible to start the cutting procedure. The heart ventricle was placed on the Petri dish on ice (Fig. 1, step 3), tissue blocks 8 mm ×8 mm were dissected by making incisions through the full thickness of the ventricular wall with a single edge steel blade (Fig. 1, step 4). Ventricle cubes were placed on top of the agarose cushion with the epicardium facing down, inside a 3D printed mold. Embedding solution (5–8 ml) was gently poured on top with a Pasteur pipette (Fig. 1, steps 5–6). Once solidified, the embedded tissue cube was extracted from the ring and squared on one side to orientate the tissue on the specimen holder and facilitate the alignment with the blade (Fig. 1, step 7). The embedded tissue was then glued using cyanoacrylate glue onto the specimen holder, with the epicardium face up. The vibratome’s blade was carefully aligned to the top of the cube, setting the slicing start point. Next, 400–500 µm thick slices were cut (Fig. 1, step 8). During cutting, the sample was constantly submerged in cold and oxygenated cutting/recovery solution. After cutting, slices were incubated for at least 30 min in cutting/recovery solution at room temperature in the recovery bath (Fig. 1, step 9) before proceeding to culture or further analysis (Fig. 1, steps 10–11).

Tissue culture

Slices were cultured epicardium-up on 8-mm-high Polydimethylsiloxane (PDMS) (SYLGARD 184) pillars cast at the bottom of a 100 mm petri dish (Fig. 1, step 10). Slices were held in places using entomology pins (A1 − 0.14 × 10 mm, Watkins & Doncaster). For static culture, the air–liquid interface was achieved by carefully adding culture medium (Medium 199 + 1X ITS Liquid Media Supplement + 1% Penicillin/Streptomycin Penicillin-Streptomycin + 10 mM of BDM -all Sigma-Aldrich) to leave the epicardium exposed to the atmosphere. This culture system maximized the contact between the myocardium and the culture medium. For epicardial cell reactivation experiments, the culture medium was supplemented with 100 ng ml−1 of Tβ4 (Human Thymosin beta 4 peptide, Abcam). For the dynamic culture, a custom-designed 3D printed adapter was inserted between the Petri dish and its lid providing inlet/outlet connection to the BioFlo 120 (Eppendorf) control station which provided a real-time feedback regulation of the medium pH at 7.4, oxygen saturation at 21% and provided a continuous flow rate of 4 ml/min. The level of the medium was precisely determined by the high of the inlet/outlet ports, maintaining a constant air–liquid interface. Slices were cultured in an incubator with humidified air at 37 °C and 5% CO2 for up to 48 h.

Live staining

Slices were incubated at room temperature with 10 μM Calcein AM cell-permeant dye (Invitrogen, Thermo Fisher Scientific) for 45 min under continuous shaking. Following washes, confocal images were collected from three random fields of each slice using a ×10 objective on a Nikon Eclipse Ti A1-A confocal laser scanning microscope. Z-stack confocal images were generated from pictures taken at 5–10 µm intervals, 1024 × 1024 pixels, from 7 to 30 sections. The percentage of area stained was measured on maximum intensity projection images using ImageJ. Cell circularity was assessed on the epicardial cell-covered areas on maximum intensity projection images of the slices stained with calcein AM, using the ImageJ BioVoxxel plugin.

Slice morphology

Slices were fixed in 4% PFA (Paraformaldehyde, Santa Cruz Biotechnology) overnight (o/n) at 4 °C, washed with phosphate buffer saline (PBS), and incubated overnight in 30% w/v sucrose (Sigma-Aldrich) solution in PBS. Slices were frozen embedded in OCT Compound (Agar scientific) in dry ice and cryostat sectioned longitudinally obtaining 5 µm thick sections.

Antigen retrieval was performed with microwave at 750 W for 15 min with citrate buffer (0.1 M Citric Acid, pH 6.0) or water bath at 80 °C for 30 min with tris-EDTA buffer (10 mM Tris Base, 1 mM EDTA Solution, pH 9.0) followed by permeabilization for nuclear antigens (0.1% Triton X-100 in PBS for 30 min) and blocked for 1 h at room temperature with 20% v/v Goat serum (Sigma-Aldrich) in PBS. Primary antibody incubation was performed overnight at 4 °C (WT1 1:50, E-cadherin 1:50, CD31 1:100, NG2 1:500 (water bath antigen retrieval performed) all from Abcam; Mesothelin 1:100 and Uroplakin IIIB 1:200 from Novus Biologicals; α-Actinin (Sarcomeric) 1:800, α-SMA 1:400, PCNA 1:100 all from Sigma-Aldrich; Connexin 43 1:300, Vimentin 1:100 all from Thermo Fisher Scientific), followed by the appropriate Goat anti-Mouse and/or Goat anti-Rabbit Alexa Fluor (Thermo Fisher Scientific) secondary antibody 488 and/or 567 diluted 1:200 for 1 h at 37 °C, and nuclei staining with DAPI (4′,6-diamidino-2-phenylindole, Merck) for 10 min at room temperature. Incubation with 0.1% Sudan Black (Sudan Black B, Santa Crus Biotechnology) in 70% ethanol w/v for 30 min at room temperature was performed to reduce tissue autofluorescence. Slides were then mounted in Fluoromount G (Invitrogen eBioscience Fluoromount G, Thermo Fisher Scientific) and imaged with Nikon Eclipse Ti A1-A confocal laser scanning microscope. Quantifications were performed on 3–7 random fields using ImageJ on 10x images.

In situ detection of apoptosis and proliferation

For the detection of cell death in situ, we used the ApopTag kit (Merck) on 5 µm thick cryosections of fixed epicardial slices. Briefly, after rinsing the sections in PBS, equilibration buffer was applied on the specimens for 10 min at room temperature. TdT enzyme incubation was performed for 1 h at 37 °C, followed by an anti-digoxigenin conjugate solution for 30 min incubation at room temperature. Nuclei were counterstained with DAPI for 10 min at room temperature. Incubation with 0.1% w/v Sudan Black (Sudan Black B, Santa Crus Biotechnology) in 70% ethanol for 30 min at room temperature was performed to reduce tissue fluorescence background. Negative control was performed by omitting the TdT enzyme in the first incubation. As a positive control we used the same tissue sections pretreated with DNase I 3U/ml for 15 min at room temperature.

The proliferation of cells was evaluated by immunohistochemistry staining for proliferating cell nuclear antigen (PCNA). After antigen retrieval, accomplished with microwave at 750 W for 15 min with citrate buffer (0.1 M Citric Acid, pH 6.0), blocking of unspecific binding of the primary antibody was performed by incubating the slides with 20% Goat serum (Sigma-Aldrich) in PBS for 1 h at room temperature. Incubation with anti-PCNA Antibody clone PC10 (Merck), diluted at 1:100 was carried overnight at 4 °C. Signal detection was provided by goat anti-mouse Alexa Fluor secondary antibody 488 incubation, diluted at 1:200, for 1 h at 37 °C. Nuclei were stained with DAPI for 10 min at room temperature. Incubation with 0.1% Sudan Black (Sudan Black B, Santa Crus Biotechnology) in 70% ethanol (w/v) for 30 min at room temperature was performed to reduce tissue autofluorescence.

In both protocols, tissue slides were then mounted in Fluoromount G (Invitrogen eBioscience Fluoromount G, Thermo Fisher Scientific) and imaged with Nikon Eclipse Ti A1-A confocal laser scanning microscope. Quantifications were performed on 3-7 random fields using ImageJ on 10x images.

Gene expression analysis

RNA was extracted using Promega reliaprep RNA Miniprep System (Promega) from homogenate tissues, and reverse-transcribed using QuantiTect Reverse Transcription Kit (Qiagen). Real-time PCR was performed on QuantStudio 7 Flex Real-Time PCR System (Applied Biosystems) using QuantiTect SYBR Green PCR Kit (Qiagen) and the primers in Supplementary Table 1 and results were normalized to the house-keeping gene β-2 microglobulin (B2M).

Statistical analysis

Measurements were taken from distinct samples. Difference among groups were evaluated using one-way ANOVA or Kruskal–Wallis test, based on results from Shapiro–Wilk normality tests, followed by Fisher’s LSD post hoc test (GraphPad Prism 8.1.2). The difference between distributions of cell circularity was calculated with Kolmogorov–Smirnov test, using T0 as a reference distribution. Difference among groups within the same circularity score were evaluated using two-way ANOVA. A value of p < 0.05 was considered statistically significant. Data were presented as mean ± SEM.

Reporting Summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.

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